This kit exhibits excellent sensitivity as it can detect fewer than
10 target copies, performs over a broad, linear dynamic range of 6 to 7
orders of magnitude, and is compatible with most real-time PCR
instruments (Fig. 2). SYBR Green I Dye is used in this kit to detect any
double-stranded DNA that accumulates during the amplification process
and also allows melt-curve analyses. No fluorescent probes are required.
Individual kit components have been carefully formulated to obtain
optimal activity of M-MLV RT, Taq DNA Polymerase, and SYBR Green I to
allow highly sensitive and specific detection of RNA transcripts from
either total RNA or poly(A)+ mRNA. Separate tubes of the passive
reference dyes ROX™ and fluorescein are included for added convenience
to allow normalization of well-to-well variations.

Fig. 2. Real-time PCR Amplification using HotStart-IT® SYBR® Green One-Step qRT-PCR Master Mix Kit (PN 75770).
GAPDH Assay using HotStart-IT® SYBR® Green One-Step qRT-PCR Master Mix
Kit (PN 75770). Duplicate reactions were performed with human placental
total RNA as template using an ABI 7500 Fast instrument. The amount of
template ranged from 100 ng to 1 pg in an order of magnitude dilution
series. The non-specific dsDNA binding dye, SYBR Green I, was used to
detect the 122 bp amplicon and ROX was used as a passive reference dye.
The amplification process was linear over six orders of magnitude with a
correlation coefficient of 0.99 (see inset). The No Template Control
(NTC) reaction generated no measurable fluorescence.

Functional Test:HotStart-IT SYBR Green One-Step qRT-PCR Master Mix Kit is a
Tested User Friendly™ product, assuring reliable results. Release
specifications for the kit are based on the following functional assay.
Real-time qRT-PCR reactions were performed on an ABI 7500 Fast
Instrument using primers specific to a 122 bp region of the human GAPDH
gene and human total RNA as template. Product specifications require
that the correlation coefficient from a linear regression over five
orders of magnitude (10 pg to 100 ng) must be greater than or equal to
0.95.